Biomedical subjects

Jishan Li

Publications and source records attributed to Jishan Li.

6 recordsLinked to original sources

PubMed2006

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification.

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.

Biosensing Techniques↗

PubMed2006

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach.

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.

Base Pair Mismatch↗

PubMed2005

A colorimetric method for point mutation detection using high-fidelity DNA ligase.

The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.

Cell Line↗

PubMed2005

Nanogold particle-enhanced oriented adsorption of antibody fragments for immunosensing platforms.

A general design strategy for immunosensing platforms has been proposed on the basis of Nanogold particle-enhanced oriented adsorption of antibody fragments. Quartz crystal microbalance (QCM) as a model transducer was fabricated with plasma-polymerized film (PPF) of n-butyl amine and then with nanogold particles resulting in a PPF-nanogold adsorption procedure for half-IgG fragments obtained by reduction of intact immunoglobulin (IgG). Thermodynamic studies reveal that the proposed procedure is superior to the traditional oriented ones in that it created immunosurface of increased antibody surface density (amount) and antigen binding constants. Sensors produced according to the new immobilization procedure exhibit better immunosensing performances including high sensitivity, fast response rate, and favorable operational stability etc. This Nanogold particle-enhanced immobilization technique may be tailored as a promising alternative for various immunosensing platforms in solid-phase immunoassay and affinity chromatography.

Adsorption↗

PubMed2004

A plasma-polymerized film for capacitance immunosensing.

A capacitance immunosensor based on a plasma-polymerized ethylenediamine film (PPEF) has been developed. The resulting PPEF is studied with scanning electrode micrograph (SEM), IR reflection spectrum and cyclic voltammetry. SEM and IR reflection spectrum showed that the plasma-polymerized film (PPF) formed on the gold electrode surface is quite homogeneous, flat, nonporous and contains plenty of free-reacted -NH2. Moreover, cyclic voltammetry showed that the hexacyanoferrate redox reactions were blocked well by the formed PPF, that is to say, the formed PPF has excellent insulating characteristics. To investigate its applicability for capacitive immunosensing, goat-anti-human IgG antibody (IgGAb) was coupled to the PPF-coated gold electrode surface via glutaraldehyde (GA) to form an immunoglobulin G (IgG) probe. Alternating current (ac) impedance and capacitance measurement were used in the immunoassay. The experiment results show that the PPEF is applicable to form insulating layer of capacitive immunosensors.

Biosensing Techniques↗

PubMed2004

A piezoelectric immunoagglutination assay for Toxoplasma gondii antibodies using gold nanoparticles.

The serologic detection of anti-Toxoplasma gondii immunoglobulins plays a key role in the clinical diagnosis of Toxoplasmosis. In this paper, a simple, rapid and highly sensitive agglutination-based piezoelectric immunoassay has been firstly developed for directly detecting anti-T. gondii immunoglobulins in infected rabbit serum (IRS) and infected rabbit blood (IRB). The proposed technique is based on that the specific agglutination of antigen-coated gold nanoparticles, averaging 10nm in diameter, in the presence of the corresponding antibody causes a frequency change that is monitored by a piezoelectric device. In contrast to the commonly used piezoelectric assays, it possesses an attractive advantage in that the immobilization of antibody or antigen on the crystal is unnecessary. Use of a newly prepared sensing probe which was modified by a plasma-polymerized film (PPF) of n-butyl amine and further by a heparin layer resulted in a response-enhanced immunoagglutination and a high compatibility of the probe with biological samples. An appropriate reagent consisting of 1% normal rabbit serum (NRS) and 0.1% bovine serum albumin (BSA) for diluting the analytes were verified in counteracting the background interference of assay. Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination rate and sensitivity enhancer was investigated in detail. It is found that the developed immunoagglutination assay system is sensitive to dilution ratio of anti-T. gondii antibody as low as 1:5500. Analytical results of several specimens obtained using the developed technique are in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting anti-T. gondii antibodies in the clinical diagnosis.

Agglutination Tests↗